This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. O-GlcNAcylation is likely to play an important role in diabetes pathology. The liver and the pancreas are the two main sites that regulate glucose levels. Preliminary work from other laboratories suggests that glucose levels modulate the abundance of O-GlcNAc modifications on proteins from hepatic cancer cells (Taylor, RP et al. JBC, 6050-7 2008.). Similarly, the _ islets of the pancreas were shown to contain high amount of O-GlcNAc modified proteins and, according to Northern blots, these cells are believed to express the highest OGT levels (Hanover JA, Arch. Bioch. Biophys. 38-45, 1999). These studies were based on immunostaining, Western or Northern blot analyses. Specific O-GlcNAcylation sites were not identified;the identity of many O-GlcNAc modified proteins remains unknown. The aim of the current project is to identify biologically relevant O-GlcNAc modification sites in pancreatic and hepatic cells. Pertinent cell lines, such as Min6 or HepG2, are grown in culture and exposed to different physiological glucose concentrations. Peptides obtained from whole cells are then subjected to O-GlcNAc enrichment and chromatographic fractionations. LC MS/MS analysis is then performed using electron transfer dissociation on an Orbitrap instrument. This will lead to the identification of O-GlcNAc modified proteins and the assignment of specific O-GlcNAc sites. It is anticipated that this study will provide insight into the protein set critical to the regulation of blood glucose levels. Ultimately, we intend to perform relative quantitation of specific O-GlcNAc peptides, to gain a better understanding of the response to glucose in both the liver and the pancreas.